The thymidine kinase gene of Herpes Simplex Virus is contained within a 3,400 base pair DNA fragment isolated by Bam HI restriction endonuclease digestion of viral DNA. The expression of this isolated DNA fragment can be demonstrated by two distinct assays. When the 3,400 base pair Bam HI fragment is introduced into thymidine kinase deficient mouse L-cells by DNA mediated transformation, thymidine kinase positive cell clones are obtained. These transformant cell clones express the HSV thymidine kinase enzyme. When the 3,400 base pair thymidine kinase DNA fragment is microinjected into Xenopus oocytes, the authentic gene product is expressed. These two assays for thymidine kinase gene function will allow a correlation to be made between specific DNA sequences and specific mechanisms involved in thymidine kinase gene expression. In order to accomplish this goal, the following preliminary experiments will be conducted. First, the complete DNA sequence of the HSV thymidine kinase gene will be resolved. Second, the regions of the gene code for thymidine kinase messenger RNA will be elucidated. Third, it will be determined if the thymidine kinase gene is interrupted by intervening DNA sequences. Fourth, the oocyte assay system will be analyzed in detail to determine which mechanisms involved in thymidine kinase gene expression function accurately and efficiently. Upon completion of these objectives, specific DNA sequences of the thymidine kinase gene will be altered systematically by mutagenesis in vitro. Variant thymidine kinase genes will be characterized by DNA sequence analysis and tested for expression in both of the functional assay systems. The results of these studies should provide a framework for understanding the mechanisms used by normal animal cells in the expression of structural genes. Such information may provide a basis for asking specific questions relevant to the abnormal expression of genes as a consequence of specific genetic diseases and cancer.